CN104328075B - Bacillus subtilis strain capable of killing algae and application thereof - Google Patents
Bacillus subtilis strain capable of killing algae and application thereof Download PDFInfo
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- CN104328075B CN104328075B CN201410624723.9A CN201410624723A CN104328075B CN 104328075 B CN104328075 B CN 104328075B CN 201410624723 A CN201410624723 A CN 201410624723A CN 104328075 B CN104328075 B CN 104328075B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
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- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
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- Medicinal Chemistry (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a bacillus subtilis strain capable of killing algae and application thereof. The strain is named (Bacillus subtilis) BLS1 and was collected in China Center for Type Culture Collection in Wuhan University on December 18, 2012, with a CCTCC No. of M2012534. The strain is capable of effectively killing harmful algae and has an obvious killing effect on microcystis aeruginosa which is most harmful to the body of fresh water so that algal bloom and red tide can be relieved and avoided in time. The application method of the strain disclosed by the invention is simple; the strain can be made into powder or tables according to a conventional method and can be put into the body of water anytime as required; in case of no red tide or algal bloom, the strain can be put into the body of water to balance floras in the body of water; the strain is safe to animals bred in the body of water, is mild in fermentation condition and can be put into culture enlargement easily.
Description
Technical field
The invention belongs to microorganism field, the more particularly, to one plant bacillus subtilises with molten algae ability and its application.
Background technology
In ocean and poisons in freshwater, due to the aggravation of Eutrophic Extent, algalbloom forms wawter bloom or red tide,
Thus impact and the physicochemical property changing water body, and can directly or indirectly cause the mortality of weed eater.Traditional controls
Reason method such as machinery removes algae, high-frequency electromagnetic pulse and using the method such as herbicide, is subjected to cost and Environmental security sex chromosome mosaicism
Restriction Deng factors.By contrast, Biological control technical costss are low, safety is good, and wherein algae-lysing bacterium is higher because of it
Except algae effect, become a new direction preventing and treating wawter bloom and red tide.
Algae-lysing bacterium (algae-lysing bacteria) is that a class suppresses algal grown in direct or indirect mode, or
Kill algae, the general designation of the antibacterial of dissolving frustule, as a weight of biotic population 26S Proteasome Structure and Function in aquatic ecosystem
Want ingredient, to the control of wawter bloom and red tide, maintain the balance of algae bio amount to have very important effect, they are to swimming
The dissolution of plant is also likely to be a key factor adjusting primary productivity in aquatic ecosystem.Wawter bloom and red tide
Suddenly withering away may be relevant with the infection of algae-lysing bacterium.Algae-lysing bacterium, as the biology of wawter bloom and red-tide control, causes domestic
The concern of many scholars outward, Abroad in Recent Years constantly isolates new algae-lysing bacterium.Comparatively, domestic report is less, and nearly ten
It is nearly at dead states for many years.
Algae-lysing bacterium can be used for the preventing and treating to the wawter bloom that the harmful algaes such as cyanophyceae cause it has been reported that such as Guo Ji etc. is from too
Lake is separated to the bacillus cereuss having algae-lysing to microcystic aeruginosa；Liu Jing etc. is also separated in the pond of Guangzhou eutrophication
There are Bacillus cercuses and Bacillus pumilus of molten algae function etc. to microcystic aeruginosa and Anabaena Flos-aquae.
The model of action of algae-lysing bacterium is generally divided into two kinds：One is direct molten algae, i.e. direct attack host, and it needs antibacterial
In molten frustule directly contact, or even intrusion frustule.Shilo M reports slime bacteria (Myxobacter) earliest to cyanophyceae
Direct dissolution, history is also direct molten algae along the bacterial strain of the discoveries such as jade.Two is molten algae indirectly, i.e. indirect attack host is main
Antibacterial to be included competes limited nutrition or the molten algae of the extracellular material of bacterial secretory with algae.Wherein secrete extracellular material molten algae document report
More, it is the main effect model of algae-lysing bacterium.Algae-lysing bacterium can pass through to discharge specificity or nonspecific extracellular material,
As protein, azanol, antibiotic, polypeptide, aminoacid etc. kill frustule.That reports indirect algae-lysing mainly has Peng Chao, Lee
Sun-og and Imamura etc..
At present, domestic and international algae-lysing bacterium is generally separated from the lake that wawter bloom or red tide occur because of eutrophication and ocean,
It is mostly gram negative bacteria.Because algae-lysing bacterium ratio ratio present in natural water is relatively low, therefore use conventional methods
Need to concentrate a large amount of water samples, time-consuming and is difficult to obtain preferable algae-lysing bacterium, far apart with practical implementation in future.Cause
This, find algae-lysing bacterium separation source, realizes in the short time bacterial strain needed for separation screening obtains, the further investigation to algae-lysing bacterium and
Application is significant.
Microcystic aeruginosa most serious of all is endangered in poisons in freshwater, in aquaculture process, if algal grown is excessive,
Water body overrich, easily causes aquatic animal to raise the nose above water to breathe, in addition suffocate, death etc..
Content of the invention
For above-mentioned prior art, the present invention provides one plant of bacillus subtilis with molten algae ability and its application, leads to
Cross screening to obtain one plant there is molten algae performance antibacterial the early stage contrived experiment applied, for developing the micro- life of biological treating cyanophyceae
State preparation provides platform.
To achieve these goals, the present invention adopts the following technical scheme that：
One plant of bacillus subtilis with molten algae ability, is named as bacillus subtilises (Bacillus subtilis)
BLS1, depositary institution：China typical culture collection center (CCTCC)；Preservation address：Wuhan, China, Wuhan University；Postcode：
430072；Preservation date：On December 18th, 2012；Deposit number：CCTCC NO：M 2012534.
The strain bio characteristic of described bacillus subtilises is as follows：Bacterial strain CCTCC M2012534 direct rod shape, single, bud
Life or closely middle life in spore, Gram’s staining is positive, and colonial morphology is circle, and opaque, aerobic bacteria anaerobism does not grow, peroxidating
Hydrogen enzyme, citrate, D-Glucose, Mannitol, casein, Starch Hydrolysis, nitrate reduction, D- xylose, arabinose, V-P are anti-
Should, sucrose react for the positive, indole, tyrosine, phenylalanine deaminase are reacted for feminine gender, and mass fraction is 2%NaCl, quality
Fraction be 5%NaCl, 30 DEG C and 50 DEG C all can grow, 5 DEG C do not grow, 28 DEG C of optimum temperature, optimal initial pH be 7.2.
A kind of microbial inoculum, active component is bacillus subtilises CCTCC M 2012534, a kind of powder or tablet, by described
Microbial inoculum is prepared from.
Described bacterial strain and/or described microbial inoculum and/or described powder and/or described tablet, answering in killing harmful algae
With described harmful algae is one of microcystic aeruginosa or chlorella or two kinds；Application in preventing and treating wawter bloom and red tide；?
The application in water body environment is administered in aquaculture.
The cultural method of described bacillus subtilises CCTCC M2012534, comprises the following steps：
(1) strain：Bacillus subtilises (Bacillus subtilis) BLS1, CCTCC M2012534；
(2) slant culture：Strain in step (1) is inoculated on solid slant culture base, in 32 DEG C～40 DEG C conditions
Lower culture 20h～28h；
(3) first order seed culture：Take cultured inclined-plane, aseptically inoculate, take two rings in 50mL with inoculating loop
In～100mL seed fluid nutrient mediums of saccharomycete, under the conditions of 32 DEG C～40 DEG C, shaking table culture 14～18h, rotating speed is 200rpm, is obtained
Primary seed solution；
(4) amplification culture：With 5% inoculum concentration, primary seed solution is connected to 500mL～1000mL seed fluid nutrient mediums of saccharomycete
In, under the conditions of 32 DEG C～40 DEG C, shaking table culture 14～18h, rotating speed is 200rpm, prepared secondary seed solution；
(5) fermentor cultivation：With 3% inoculum concentration, secondary seed solution is connected in liquid fermentation medium, in 20 DEG C～
Under the conditions of 37 DEG C, rotating speed is 200rpm, after culture 28h～35h, when spore rate reaches more than 90%, collects fermentation liquid, obtains final product
Microbial inoculum；
(6) add 4～8% (mass percent) corn starch in fermentation liquid, be spray-dried immediately, prepared mycopowder.
Described in above-mentioned steps (3), (4), seed fluid nutrient mediums of saccharomycete formula is：Glucose 20g/L, peptone 10g/L, ferment
Female cream 5g/L, during use, adjusts pH to 6.5～7.5, and sterilize under the conditions of 120 DEG C 25min；The described solid slant culture of step (2)
Base is to add the agar powder that mass fraction is 1.5～2.0% in above-mentioned seed fluid nutrient mediums of saccharomycete；Described in step (5), liquid is sent out
Ferment culture medium prescription is：Glucose 20g/L, peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L, during use, adjust pH to
Sterilize under the conditions of 6.5～7.5,120 DEG C 25min.
Mycopowder is pressed into tablet；Described bacterial strain and/or the application of described microbial inoculum and/or described powder and/or described tablet
Method, is directly invested in water.
Beneficial effects of the present invention：
1st, the present invention endangers microcystic aeruginosa the most serious as pattern algae kind using in poisons in freshwater, filters out one plant of energy
The algae-lysing bacterium of enough effectively preventing and treating algae harm, the bacillus subtilises CCTCC M2012534 of screening can effectively kill
Evil algae, to endangering microcystic aeruginosa effect the most serious in poisons in freshwater substantially, timely alleviate and avoid wawter bloom and
Red tide phenomenon.
2nd, the strain application process of the present invention is simple, conventionally makes powder or tablet, as needed with
When be invested in water body.
3rd, in the case that red tide or wawter bloom do not occur, throw in this strain in water body, the flat of water body flora can be maintained
Weighing apparatus.
4th, by, being learnt, the strain of the present invention is safe to aquifer cultivation animal to the evaluation of water body animal safety,
No matter being artificial challenge or manual injection's test, all bad phenomenon in water body animal, so strain of the present invention is a kind of
It is applied to the reliable strain of freshwater aquiculture.
5th, the strain fermentation mild condition of the present invention is it is easy to amplification culture.
Brief description
One plant of bacillus subtilis with molten algae ability, is named as bacillus subtilises (Bacillus subtilis)
BLS1, depositary institution：China typical culture collection center (CCTCC), preservation address：Wuhan, China, Wuhan University, postcode：
430072, preservation date：On December 18th, 2012, deposit number：CCTCC NO：M 2012534；
Fig. 1 is the preliminary judgement result of the bacterial strain algicidal effect of the present invention, and No. 0 is control bottle, and 1～No. 3 is from Tai'an
The eutrophication fresh water water sample of Dai Temple collection, 4～No. 6 is the eutrophication fresh water water sample gathering from Tai'an South Lake, 7～No. 10
It is the eutrophication fresh water water sample from the collection of Tai'an East Lake；
Fig. 2 is the result of determination of the single strain algicidal effect of the present invention, and a, b, c, d bottle is the 36 plants of individual plants holding successively
Bacterium；
Fig. 3 is the algicidal effect of bacterial strain 1-17；
Fig. 4 is the molten algae culture effect of bacterial strain 1-17, and wherein four conical flasks are control bottle, bacteria suspension from left to right successively
Concentration is M1-108Cfu/mL bottle, bacteria suspension concentration are M2-107Cfu/mL bottle, bacteria suspension concentration are M3-106Cfu/mL bottle；
Fig. 5 is the algicidal effect of bacterial strain BLS1；
Fig. 6 is the molten algae culture effect of bacterial strain BLS1, and wherein four conical flasks are control bottle, bacteria suspension from left to right successively
Concentration is M1-108Cfu/mL bottle, bacteria suspension concentration are M2-107Cfu/mL bottle, bacteria suspension concentration are M3-106Cfu/mL bottle；
Fig. 7 is the algicidal effect of bacterial strain 2-3；
Fig. 8 is the molten algae culture effect of bacterial strain 2-3, and wherein four conical flasks are control bottle, bacteria suspension from left to right successively
Concentration is M1-108Cfu/mL bottle, bacteria suspension concentration are M2-107Cfu/mL bottle, bacteria suspension concentration are M3-106Cfu/mL bottle；
Fig. 9 is the algicidal effect that bacterial strain 1-17 and bacterial strain BLS1 compounds；
Figure 10 is the molten algae culture effect that bacterial strain 1-17 and bacterial strain BLS1 compounds, and wherein four conical flasks are from left to right successively
Be control bottle, bacteria suspension concentration be M1-108Cfu/mL bottle, bacteria suspension concentration are M2-107Cfu/mL bottle, bacteria suspension concentration are M3-
106Cfu/mL bottle；
Figure 11 is the algicidal effect that bacterial strain 2-3 and bacterial strain BLS1 compounds；
Figure 12 is the molten algae culture effect that bacterial strain 2-3 and bacterial strain BLS1 compounds, and wherein four conical flasks are from left to right successively
Be control bottle, bacteria suspension concentration be M1-108Cfu/mL bottle, bacteria suspension concentration are M2-107Cfu/mL bottle, bacteria suspension concentration are M3-
106Cfu/mL bottle；
Figure 13 is the safety evaluatio to chlorella for the bacterial strain 1-17；
Figure 14 is the safety evaluatio to chlorella for the bacterial strain BLS1；
Figure 15 is the safety evaluatio to chlorella for the bacterial strain 2-3；
Figure 16 is bacterial strain BLS1 growth curve count plate；
Figure 17 is bacterial strain BLS1 growth curve OD value；
Figure 18 is bacterial strain 1-17 growth curve count plate；
Figure 19 is bacterial strain 1-17 growth curve OD value；
Figure 20 is the application effect of bacterial strain BLS1.
Specific embodiment
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
1 materials and methods
1.1 test material
1.1.1 algae kind：(both of which is purchased from Inst. of Hydrobiology, Chinese Academy of Sciences algae for microcystic aeruginosa, chlorella
Plant storehouse).
1.1.2 strain：Separate from the poisons in freshwater of Tai'an city (East Lake, South Lake, Dai Temple) eutrophication.
1.1.3 experimental animal：Carassius auratuss are purchased from Tai'an five Horse market field；Test is purchased from Tai'an hot spring fry factory with Cyprinus carpio.
1.1.4 culture medium：Microcystic aeruginosa culture medium：I.e. No. 10 culture medium of Zhu Shi, pH=7.1；Bead algae culture medium：I.e.
F/2 culture medium, pH=7.0；Bacteria culture media：I.e. nutrient agar；Bacillus culture medium；Each culture medium composition is shown in Table
1.
Table 1 culture medium constituent
1.2 testing equipment：Illumination box, standard incubator, low speed centrifuge, spectrophotometer, optical microscope, water
Race's case, shaking table, superclean bench, pressure cooker etc..
1.3 test method
1.3.1 the culture of algae kind
1.3.1.1 the activation of microcystic aeruginosa：Before test, first by buy microcystic aeruginosa pure strain in 2000Lx, 25
Quiescent culture in illumination box under the conditions of DEG C, once (all aseptically carries out) every the artificial shake of 3h, continues pre-
Culture one week, and microscopy has or not miscellaneous algae and algal grown situation, such as reaches synchronization and well grows the algae kind so that it may as test
Liquid.
1.3.1.2 the activation of chlorella：The same microcystic aeruginosa of activation method of chlorella, the activation of algae kind and amplification culture are adopted
Culture medium is F/2 culture medium.
1.3.2 algae solution preparation
Aseptic take algae solution, 4000rpm is centrifuged 10min, abandoning supernatant, frond liquid 15mg/L NaHCO3Washing two
All over rear as inoculation algae solution.In terms of each triangular flask 50mL, after inoculation, the initial density of microcystic aeruginosa is OD value (450nm)
0.005(1.26×105cfu/mL).
1.3.3 experimental animal feeding
Test is purchased from Tai'an five Horse market field, average weight (160 ± 15) g with Carassius auratuss, temporarily supports in indoor glass aquarium
(length, width and height are respectively：0.9m, 0.5m, 0.5m) in, effective water volume is 180L.Using the tap water of abundant aeration dechlorination as following
Ring water source, water temperature controls in (23 ± 1) DEG C, and 24h continues aeration aerating；Day feeding volume is the 1.5% of fish body weight, early, middle and late fixed
When feed.After the adaptive training of 14d, test fish is randomly divided into three test group, every group of 15 tails, test period by body weight
For 21d.
1.3.4 the separation of algae-lysing bacterium and screening
1.3.4.1 the separation of wild algae-lysing bacterium
(1) gather water sample from the water body of testing site with hydrophore, water sample is filtered with 0.45 μm of fibrous filter membrane, by 10mL filtrate
40mL is added to cultivate in the microcystic aeruginosa culture fluid of 5d, mix homogeneously, by the condition of culture culture 7d of microcystic aeruginosa activation
Afterwards, using the algae solution of yellow as the material of algae-lysing bacterium, take yellow algae solution by 10-1、10-2、10-3、10-4、10-5、10-6Gradient is dilute
Release, flat board is coated with, line separates, 37 DEG C of culture 48h, obtain single bacterium colony.
(2) single bacterium colony is inoculated in nutrient agar, 37 DEG C of shaken cultivation 24h, takes 10mL bacterium solution to add
In 100mL microcystic aeruginosa liquid, after the condition of culture culture 7d of microcystic aeruginosa activation, by obvious for algae solution yellow, molten algae effect
Really best diluted sample, flat board is coated with, and line separates, and obtains single bacterium colony, repeats aforesaid operations to verify the bacteriolyze of bacterial strain
Effect.
(3) by inoculation stable for algicidal effect in nutrient agar, 37 DEG C of shaken cultivation 24h,
4000rpm, centrifugation 10min is configured to 1 × 10 after collecting bacterium mud, the brine being 0.75% with mass fraction three times8、
1×107、1×106The bacteria suspension of cfu/mL, takes 5mL bacteria suspension to be added in the middle of 50mL microcystic aeruginosa liquid respectively, through 7d prison
Survey the change (measuring using heat ethanol methodses) of chlorophyll content, chlorophyll content is reduced to preferable group of algicidal effect.
Method for measuring chlorophyll content (heat ethanol methodses)：Take 3mL algae solution in 10mL centrifuge tube, 4000rpm is centrifuged 10min
Abandon the ethanol solution that supernatant adds 5mL volume fraction to be 90% afterwards, test tube is placed in extraction 5min in 75 DEG C of water-baths, takes it to carry
Liquid is taken to be measured.Absorption values at itself 665nm and 750nm are read using spectrophotometer, is added thereto to 1mol/L
Hydrochloric acid, again reads off the absorption values at itself 665nm and 750nm after 8min.Calculate chlorophyll content using formula：C
(chla)=27.3 (Ea Eb) Ve/V, in formula, C (chla) is the content μ g/L of water sample Determination of Chlorophyll a, before Ea is for extracting solution acidifying
The difference of the optical density at wavelength 665nm and 750nm, Eb for the optical density at wavelength 665nm and 750nm after extracting solution acidifying it
Difference, Ve is the cumulative volume (5mL) of extracting solution, and V is the volume of water sample (3mL) of sucking filtration.
(4) compounded filtering out the best algae-lysing bacterium of algicidal effect two-by-two with 2.5mL with other strains：2.5mL
Ratio add, be finally all configured to concentration 1 × 108、1×107、1×106Cfu/mL, selects the preferable Compound bacterium of algicidal effect
Kind.
1.3.5 the identification of algae-lysing bacterium
The identification of algae-lysing bacterium is carried out by the way of Physiology and biochemistry identification is combined with 16S rDNA identification.Physiology and biochemistry
The preparation of test used medium is shown in《Common bacteria system identification handbook》(east show pearl, the calendar year 2001 first edition) and《Microbiology
Experiment》(Shen Ping, fourth edition in 2007).
1.3.5.1 the amplification of 16S rDNA and sequence analysis
Purpose inoculation cultivates 20h in fresh bacillus culture medium, and the test kit using Tiangeng company extracts
Thallus DNA, and it is carried out with 16S rDNA sequence amplification.The primer is universal primer：Forward primer 5 '-
Agagtttgatcctggctcag-3 ' downstream primer 5 '-aaggaggtgatccagccgca-3 ', PCR reaction system (50 μ L)
For:DNTP Mixture 4 μ L (archaeal dna polymerase containing Taq and dNTP etc., Tiangeng biochemical technology company limited), 5 × Primer
STAR Buffer 10 μ L, each 0.6 μ L of upstream and downstream primer, template DNA 1 μ L, enzyme 0.5 μ L, ultra-pure water 33.3 μ L.PCR expands journey
Sequence is 95 DEG C of denaturations 5min, 94 DEG C of degeneration 1min, 59 DEG C of annealing 15s, 72 DEG C of extension 45s, 30 circulations, 72 DEG C of extensions
10min.PCR primer send Beijing Bo Shang Bioisystech Co., Ltd to carry out sequencing.
1.3.5.2 Physiology and biochemistry
Testing index：Anaerobic growth, catalase, citrate, D-Glucose, indole, Mannitol, casein, cheese ammonia
Acid, Starch Hydrolysis, nitrate reduction, D- xylose, arabinose, growth temperature (5 DEG C, 30 DEG C, 50 DEG C), NaCl (mass fraction
2%th, mass fraction 5%), phenylalanine deaminase, V-P reaction, sucrose.Assay method is shown in《Microbiology Experiment》(Shen Ping,
Fourth edition in 2007).
1.3.6 algicidal effect certainty evaluation
By the algae-lysing bacterium identifying inoculation in bacillus culture medium, 37 DEG C of shaken cultivation 24h, 4000rpm,
Centrifugation 10min is configured to 1 × 10 after collecting bacterium mud, the brine being 0.75% with mass fraction three times8、1×107、1
×106The bacteria suspension of cfu/mL, takes 5mL bacteria suspension to be added in the middle of 50mL microcystic aeruginosa liquid respectively.Observe algicidal effect examination
Cycle of testing is 7d, and multiple three times of the certainty evaluation counterpoise of each test strain and compounding bacterial strain algicidal effect, to obtain definitiveness
Result.Observation index：Sediments microscope inspection frustule situation of change, perusal algae solution color change, algae solution chlorophyll content
Measure (adopting heat ethanol methodses).
1.3.7 the safety evaluatio of algae-lysing bacterium
1.3.7.1 cultivated animals safety evaluation
(1) Experimental infection：
Test takes healthy Carassius auratuss 60 tail with Carassius auratuss after 14d temporarily supports and observes, and is divided into 4 groups, every group of 15 tails, and wherein three groups are
Test group, one group is matched group.Add the algae-lysing bacterium isolated in test group water body, so that the bacteria containing amount of water body is reached
108Cfu/mL, carries out Experimental infection.Observe the activity situation of Carassius auratuss, morbidity and death condition in course of infection.
(2) manual injection's test：
The thalline of culture 24h is collected by centrifugation, washs and be configured to for 0.75% physiological saline solution through mass fraction
108The algae-lysing bacterium suspension of cfu/mL, takes healthy Carassius auratuss 60 tail, is divided into 4 groups, every group of 15 tails, and wherein three groups is test group, one group
For matched group.The algae-lysing bacterium suspension through above-mentioned process for the test group Carassius auratuss every tail intraperitoneal inoculation 0.1mL, matched group injects 0.1mL
Physiological saline solution.Observe the activity situation of Carassius auratuss, morbidity and death condition after injecting.After injection, 15d analyses Carassius auratuss, sees
Examine internal organs and have or not pathological change.
1.3.7.2 pattern algae kind safety evaluation
With chlorella for pattern algae kind, single bacterium colony is inoculated in nutrient agar, 37 DEG C of shaken cultivation 24h,
Take 10mL bacterium solution to add in 100mL chlorella algae solution, after above-mentioned condition of culture culture 7d, observe frustule situation of change, algae
Liquid yellowing, the change of algae solution chlorophyll content.
1.3.8 the research of algae-lysing bacterium fermentation condition
1.3.8.1 the determination of optimum culturing temperature：By the inoculation identifying in fluid medium, respectively 28
DEG C, 30 DEG C, 34 DEG C, at a temperature of 37 DEG C, 160rpm shaking table culture 48h, measure the viable count in fermentation liquid.
1.3.8.2 the determination of optimal initial pH value：By the inoculation identifying to original ph be respectively 6.6,6.8,
7.0th, 7.2,7.4,7.6 fluid medium, the optimum culturing temperature determining by test 1.3.8.1,160rpm shaking table culture
48h, measures the viable count in fermentation liquid.
1.3.8.3 the determination of optimal ventilation amount：By the bacterial strain identifying in different sample-loading amounts (the bottled sample of 500mL triangle
50mL, 100mL, 150mL, 200mL) and shaking speed (160rpm, 180rpm, 200rpm, 220rpm) under the conditions of measure thalline
The change of growing amount, measures thalline content respectively after 30 DEG C of culture 36h.
1.3.8.4 the determination of optimum inoculation amount：By the bacterial strain identifying respectively according to 4%, 6%, 8%, 10%, 12%
Inoculum concentration is inoculated, 35 DEG C, 220rpm, cultivates 48h, detection Biomass and sporulation level.
1.3.9 the mensure of algae-lysing bacterium growth curve
Growth curve of bacteria is measured using two methods of turbidimetry and viable bacteria technology law：60mL liquid culture is taken to be based on
In 250mL triangular flask, cultivated in triangular flask with the molten phycomycete culture fluid that aseptic straw accurately draws 2.5mL culture 18h, by it
It is placed in shaken cultivation in shaking table, 30 DEG C of cultivation temperature, rotating speed 160rpm.During every 2h, sampling once, is existed with 720 type spectrophotometers
Measure absorbance at 550nm, carry out count plate microscopy spore rate simultaneously.
1.3.10 the application test of algae-lysing bacterium
Test is purchased from Tai'an hot spring fry factory, average weight (55 ± 5) g with Cyprinus carpio, is divided into matched group and test group, and each 90
Tail, temporarily supports respectively in cement culturing pool (4m × 1.2m × 1.5m), and effective water volume is 4 cubes, 29 ± 0.5 DEG C of water temperature.Water
Body daily afternoon 14:00 oxygenation continues 2h, and day feeding volume is the 5% of fish body weight, and early, middle and late timing feeds, and feeds through 43d
After raising, blue-green alga bloom phenomenon in water body, finds that advantage algae is microcystic aeruginosa, secondary advantage algae is Anabaena after microscopy.Now
Putting into 200mL viable count in water body is 5 × 108BLS1 bacterium solution, respectively at 5d, 7d measure water body chlorophyll content (adopt
Heat ethanol methodses).
2 results and analysis
The screening of 2.1 algae-lysing bacterium
As shown in Figure 1, through the culture of 7d, there is aetiolation in 1,4,5, No. 6 bottles, remaining all no obvious aetiolation,
Can be determined that 1,4,5, No. 6 bottles have algae-lysing microorganism, and yellow algae solution has been carried out with strains separation and 33 plant wild mushrooms are obtained.
Have 10 in as shown in Fig. 2 36 plants of single strains (33 plants of wild mushrooms, the existing strain of 3 plants of laboratorys), 11,12,18,19,20,22,
23rd, 24,28,31,32,33,35,36 amount to 15 plants occur in that obvious aetiolation, according to the time that aetiolation occurs
Sieve to obtain 3 plants of wild algae-lysing bacterium, respectively bacterial strain 1-17, bacterial strain BLS1, bacterial strain 2-3 eventually, and strain has been carried out to this three plants of bacterium
Identification.
The identification of 2.2 algae-lysing bacterium
2.2.1 biochemical reactions qualification result
As shown in Table 2, bacterial strain 1-17 can anaerobic growth, catalase, D-Glucose, Mannitol, Starch Hydrolysis, nitric acid
Salt reduction, D- xylose, arabinose, V-P reaction, sucrose react for the positive, citrate, indole, casein, tyrosine, phenylpropyl alcohol
Propylhomoserin deamination enzyme reaction is feminine gender, does not grow in 5%NaCl, 5 DEG C and 50 DEG C, 2%NaCl and 30 DEG C of well-grown；Bacterial strain BLS1
Anaerobism does not grow, catalase, citrate, D-Glucose, Mannitol, casein, Starch Hydrolysis, nitrate reduction, D- wood
Sugar, arabinose, V-P reaction, sucrose react for the positive, and indole, tyrosine, phenylalanine deaminase are reacted for feminine gender, and 2%
NaCl, 5%NaCl, 30 DEG C and 50 DEG C all can grow, and 5 DEG C do not grow；Bacterial strain 2-3 anaerobism can grow, catalase, citric acid
Salt, D-Glucose, casein, tyrosine, Starch Hydrolysis, nitrate reduction, V-P reaction, sucrose react for the positive, indole, manna
Alcohol, D- xylose, arabinose, phenylalanine deaminase are reacted for feminine gender, 2%NaCl, 5%NaCl, 30 DEG C can grow, 5 DEG C, 50
DEG C do not grow.
2 three plants of bacterium biochemical reactions results of table
Note：" " reaction negative, "+" reacting positive.
2.2.2 sequencing result
2.2.2.1 the 16S rDNA sequencing result of three plants of bacterium is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
2.2.3 qualification result
The wild algae-lysing bacterium qualification result of table 3
Table 3 result shows, finally draws qualification result in conjunction with 16S rDNA and biochemical reactions：Bacterial strain 1-17 is starch
Liquefaction bacillus cereuss, bacterial strain BLS1 is bacillus subtilises, and bacterial strain 2-3 is bacillus thuringiensiss.
2.3 algicidal effect certainty evaluations
2.3.1 bacterial strain 1-17 algicidal effect
Table 4 bacterial strain 1-17 algicidal effect chla (μ g/L) meansigma methodss
It is from table 4, Fig. 3, Fig. 4, through the molten algae test of 7d, all on a declining curve in 1d, 2d, 3d chlorophyll content,
After 4d, erratic growth in chlorophyll content each group, and during 7d, chlorophyll content all exceeds initial value, but is less than matched group；
Yellow effect only high concentration group is slightly obvious, and remaining two groups are visible by naked eyes aetiolation.Analysis reason is possibly added the initial stage, with
The growth and breeding of bacterial strain 1-17, effect of algae restraint is obvious, but prolongation over time, algal control produced by bacterial strain 1-17
Material simultaneously is insufficient to completely inhibit algal grown, therefore occurs in that the continued growth phenomenon of later stage algae, if analyzing prolongation training
The foster time is it is possible that the result maintaining an equal level with matched group is it is considered to actual effect and the Cost Problems of practical application are it is possible to determine that bacterial strain
Although 1-17 has algicidal effect but is not the optimal bacterial strain of algicidal effect.
2.3.2 bacterial strain BLS1 algicidal effect
Table 5 bacterial strain BLS1 algicidal effect chla (μ g/L) meansigma methodss
From table 5, Fig. 5, Fig. 6, at the end of 7d, each concentration group chlorophyll content is all on a declining curve, and be below right
According to group；All obvious aetiolation in each group.Result of the test can be seen that the effect of algae restraint of bacterial strain BLS1 substantially, and whole examination
Testing the chlorophyll content in the cycle is all to reduce, illustrate bacterial strain BLS1 effect of algae restraint ideal and relatively stably it is considered to
The problems such as ageing and use cost of practical application, it has higher using value, therefore, it can judge that bacterial strain BLS1 is
There is one plant of algae-lysing bacterium of using value.
2.3.3 bacterial strain 2-3 algicidal effect
Table 6 bacterial strain 2-3 algicidal effect chla (μ g/L) meansigma methodss
As shown in table 6, Fig. 7, Fig. 8, at the end of 7d, each concentration group chlorophyll content is below matched group, but is above just
Initial value；Wherein aetiolation in only high concentration group, and remaining two groups are all visible by naked eyes aetiolation.Analysis reason possibly adds
Plus the initial stage is with the growth and breeding of bacterial strain 2-3, effect of algae restraint is obvious, but prolongation over time, and bacterial strain 2-3 produced
Anti-algal substance and be insufficient to completely inhibit algal grown, therefore occur in that the continued growth phenomenon of later stage algae, but it be highly concentrated
Degree group grows slowly, if continuing to increase usage amount it is possible that preferable effect of algae restraint, but cost also accordingly increases,
Although the actual effect of consideration practical application and Cost Problems are it is possible to determine that bacterial strain 2-3 has algicidal effect but is not algicidal effect
Optimal bacterial strain, but its algicidal effect and using value are better than bacterial strain 1-17.
2.3.4 bacterial strain 1-17 and bacterial strain BLS1 compounds algicidal effect
Table 7 bacterial strain 1-17 and bacterial strain BLS1 compounds algicidal effect chla (μ g/L) meansigma methodss
As shown in table 7, Fig. 9, Figure 10, after 7d terminates, each concentration group chlorophyll content is below matched group, but is above just
Initial value.As can be seen from the table, bacterial strain 1-17 and bacterial strain BLS1 has algicidal effect after compounding, but inconspicuous, and chlorophyll contains
Although amount is less than matched group, it is constantly in propradation, and perusal has no obvious aetiolation in addition to high concentration group,
Therefore can be determined that, it is unsatisfactory that bacterial strain 1-17 and bacterial strain BLS1 compounds algicidal effect.
2.3.5 bacterial strain BLS1 and bacterial strain 2-3 compounds algicidal effect
Table 8 bacterial strain BLS1 and bacterial strain 2-3 compounds algicidal effect chla (μ g/L) meansigma methodss
As shown in table 8, Figure 11, Figure 12, at the end of 7d, each concentration group chlorophyll content is less than initial value, and naked eyes are visible
Aetiolation obvious.Analysis reason is it may be possible to because the effect of algae restraint of two plants of bacterium serves synergism, therefore algicidal effect
Substantially, and relatively stable.
2.4 algicidal effect safety evaluatios
2.4.1 the safety evaluatio of cultivated animals
Table 9 algae-lysing bacterium is to cultivated animals safety evaluatio result
As shown in table 9, the three plants of algae-lysing bacterium got are all safe to cultivated animals, no dead within the test period
Phenomenon, ingests normal, obvious bad phenomenon also in manual injection's test.
2.4.2 the safety evaluatio of pattern algae kind
2.4.2.1 the safety evaluatio to chlorella for the bacterial strain 1-17
The safety evaluatio to chlorella for the table 10 bacterial strain 1-17
As shown in table 10, Figure 13, bacterial strain 1-17 has molten algae property to chlorella, does not have the spy of the molten algae of clear and definite selectivity
Point.
2.4.2.2 the safety evaluatio to chlorella for the bacterial strain BLS1
The safety evaluatio to chlorella for the table 11 bacterial strain BLS1
As shown in table 11, Figure 14, bacterial strain BLS1 has molten algae property to chlorella, does not have the spy of the molten algae of clear and definite selectivity
Point.
2.4.2.3 the safety evaluatio to chlorella for the bacterial strain 2-3
The safety evaluatio to chlorella for the table 12 bacterial strain 2-3
As shown in table 12, Figure 15, bacterial strain 2-3 has molten algae property to chlorella, does not have the feature of the molten algae of clear and definite selectivity.
The research of 2.5 algae-lysing bacterium fermentation conditions
, only to bacterial strain BLS1, bacterial strain 1-17 has carried out grinding of fermentation condition for the using value of consideration bacterial strain and algicidal effect
Study carefully.
2.5.1 temperature
The impact to viable count for table 13 temperature
Result of the test shows, bacterial strain BLS1 viable count highest under conditions of 28 DEG C, and bacterial strain 1-17 is under conditions of 34 DEG C
Viable count highest.
2.5.2pH value
The impact to viable count for the table 14pH value
As shown in table 14, bacterial strain BLS1 viable count highest under the conditions of pH7.0, bacterial strain 1-17 viable bacteria under the conditions of pH7.2
Number highest.
2.5.3 the determination of ventilation
The impact to BLS1 Biomass and spore rate for table 15 rotating speed
Result of the test shows, the impact to BLS1 for the rotating speed is little, the viable count of slow-speed of revolution 140rpm and high rotating speed 220rpm
All 7.0 × 108Cfu/g, and spore rate is with 200rpm and 220rpm highest, respectively 95% and 96%, but with low turn
The spore forming rate difference of speed is inconspicuous.
The impact to BLS1 Biomass and spore rate for table 16 liquid amount
Result of the test shows, final determination liquid amount is to fill 60mL culture medium in 250mL triangular flask.
2.5.4 the impact to BLS1 Biomass and spore rate for the inoculum concentration
The impact to BLS1 Biomass and spore rate for table 17 inoculum concentration
When inoculum concentration is 12%, spore rate is up to 92%.Reason is probably that inoculum concentration is bigger, nutrient substance in culture medium
Consume is faster, reaches that the growth platform phase is faster, is more conducive to the formation of spore.
The mensure of 2.6 algae-lysing bacterium growth curves
Table 18 bacterial strain BLS1 and bacterial strain 1-17 growth curve
As shown in table 18, Figure 16, Figure 17, Figure 18, Figure 19, bacterial strain BLS1 viable count peak occurs in 40h, bacterial strain 1-17
Viable count peak 44h.
The application test of 2.7 algae-lysing bacterium
Table 19 bacterial strain BLS1 application effect
As shown in table 19, Figure 20, water body Determination of Chlorophyll content is decreased obviously, and can see water body table from field observation result
The cyanophyceae in face is under control, and can be found that microcystic aeruginosa and Anabaena quantity significantly reduce by microscopy result, and cultivates
The phenomena of mortality in animal.This result reaches test expection, because through laboratory research, bacterial strain BLS1 proves that it has necessarily
Algicidal effect, therefore this result illustrates that bacterial strain BLS1 has the ability processing breeding water body cyanophyceae problem, and because it is raw
Thing removes algae, and processing procedure relative chemical algicide is soft, and what cultivated animals were caused stress be less, asks solving water body cyanophyceae
Cultivated animals are protected while topic.
Although the above-mentioned accompanying drawing that combines is described to the specific embodiment of the present invention, not model is protected to the present invention
The restriction enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme, and those skilled in the art are not
Need to pay the various modifications that creative work can make or deformation still within protection scope of the present invention.
Claims (10)
1. one plant of bacillus subtilis with molten algae ability it is characterised in that be named as bacillus subtilises (Bacillus subtilis) BLS1, depositary institution：China typical culture collection center (CCTCC), preservation address：Wuhan, China, Wuhan is big
Learn, preservation date：On December 18th, 2012, deposit number：CCTCC NO：M 2012534.
2. bacillus subtilises as claimed in claim 1 are it is characterised in that strain bio characteristic is as follows：Direct rod shape, single
Individual, life or closely middle life in spore, Gram’s staining is positive, and colonial morphology is circle, opaque, aerobic bacteria, and anaerobism does not grow,
Catalase, citrate, D-Glucose, Mannitol, casein, Starch Hydrolysis, nitrate reduction, D- xylose, arabinose,
V-P reaction, sucrose react for the positive, and indole, tyrosine, phenylalanine deaminase are reacted for feminine gender, and mass fraction is 2%
NaCl, mass fraction be 5%NaCl, 30 DEG C and 50 DEG C all can grow, 5 DEG C do not grow, 28 DEG C of optimum temperature, and optimal initial pH is
7.2.
3. there is the cultural method of the bacillus subtilises of molten algae ability as claimed in claim 1 it is characterised in that include with
Lower step：
(1) strain：Bacillus subtilises (Bacillus subtilis) BLS1, CCTCC M 2012534；
(2) slant culture：Strain in step (1) is inoculated on solid slant culture base, trains under the conditions of 32 DEG C～40 DEG C
Foster 20h～28h；
(3) first order seed culture：Take cultured inclined-plane, aseptically inoculate, with inoculating loop take two rings in 50mL～
In 100mL seed fluid nutrient mediums of saccharomycete, under the conditions of 32 DEG C～40 DEG C, shaking table culture 14～18h, rotating speed is 200rpm, is obtained one
Level seed liquor；
(4) amplification culture：With 5% inoculum concentration, primary seed solution is connected to 500mL～1000mL seed fluid nutrient mediums of saccharomycete
In, under the conditions of 32 DEG C～40 DEG C, shaking table culture 14～18h, rotating speed is 200rpm, prepared secondary seed solution；
(5) fermentor cultivation：With 3% inoculum concentration, secondary seed solution is connected in liquid fermentation medium, in 20 DEG C～37
Under the conditions of DEG C, rotating speed is 200rpm, after culture 28h～35h, when spore rate reaches more than 90%, collects fermentation liquid；
In above-mentioned steps (3), (4), described seed fluid nutrient mediums of saccharomycete formula is：Glucose 20g/L, peptone 10g/L, yeast
Cream 5g/L, during use, adjusts pH to 6.5～7.5, and sterilize under the conditions of 120 DEG C 25min；The described solid slant culture of step (2)
Base is to add the agar powder that mass fraction is 1.5～2.0% in above-mentioned seed fluid nutrient mediums of saccharomycete；Described in step (5), liquid is sent out
Ferment culture medium prescription is：Glucose 20g/L, peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L, during use, adjust pH to
Sterilize under the conditions of 6.5～7.5,120 DEG C 25min.
4. a kind of microbial inoculum is it is characterised in that active component is the bacillus subtilises described in claim 1.
5. a kind of powder is it is characterised in that be prepared from by the microbial inoculum described in claim 4.
6. the preparation method of powder as claimed in claim 5 is it is characterised in that add in the microbial inoculum described in claim 4
Corn starch, after addition, the mass percent of corn starch is 4～8%, is spray-dried immediately, prepared mycopowder.
7. a kind of tablet it is characterised in that by the microbial inoculum described in claim 4 or by described in claim 5 powder preparation and
Become.
8. the preparation method of tablet as claimed in claim 7 is it is characterised in that be pressed into piece by the powder described in claim 5
Agent.
9. there is described in claim 1 application in killing harmful algae of the bacillus subtilises of molten algae ability；Described harmful
Algae is one of microcystic aeruginosa or chlorella or two kinds.
10. the bacillus subtilises of molten algae ability of having described in claim 1 are preventing and treating wawter bloom and red tide, are controlling in aquaculture
Application in reason water body environment.
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| CN106148264B (en) * | 2016-07-14 | 2019-08-06 | 贝嘉美(天津)生物技术研发股份有限公司 | One plant of bacillus amyloliquefaciens with the molten algae ability enhanced and its application |
| CN107177538B (en) * | 2017-07-19 | 2019-12-06 | 扬州大学 | Marine bacillus cereus and application thereof in preventing and controlling anabaena flos-aquae |
| CN111296490A (en) * | 2020-02-20 | 2020-06-19 | 福建师范大学 | Method for inhibiting chain-like gymnodinium by coupling coagulant and algae inhibiting bacteria fermentation liquor |
| CN112266300A (en) * | 2020-12-14 | 2021-01-26 | 烟台水禾土生物科技有限公司 | Method for fermenting algae microorganism using bacillus |
| CN115895935B (en) * | 2022-08-08 | 2024-02-09 | 上海海洋大学 | Bacillus subtilis and application thereof in moss control |
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| CN101830567A (en) * | 2010-04-29 | 2010-09-15 | 南京神克隆科技有限公司 | Method and biochemical treatment agent for preventing and controlling blue-green algae |
| CN103238543A (en) * | 2013-05-20 | 2013-08-14 | 大连市水产技术推广总站 | Method for controlling large algae in sea cucumber pond on basis of biotechnology |
| CN103923850A (en) * | 2013-09-13 | 2014-07-16 | 领绿生物镇江有限公司 | Microbial preparation for degrading nitrites and inhibiting blue algae and its preparation method |
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| CN101830567A (en) * | 2010-04-29 | 2010-09-15 | 南京神克隆科技有限公司 | Method and biochemical treatment agent for preventing and controlling blue-green algae |
| CN103238543A (en) * | 2013-05-20 | 2013-08-14 | 大连市水产技术推广总站 | Method for controlling large algae in sea cucumber pond on basis of biotechnology |
| CN103923850A (en) * | 2013-09-13 | 2014-07-16 | 领绿生物镇江有限公司 | Microbial preparation for degrading nitrites and inhibiting blue algae and its preparation method |
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